Injectable antibiotic formulations and use thereof

ABSTRACT

Provided herein are pharmaceutically acceptable compositions containing macrolide antibiotics, in particular azithromycin. In particular, compositions containing azithromycin with low toxicity, especially for administration to felines, are provided herein.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of priority under 35 U.S.C. §119(e)of U.S. Ser. No. 62/312,382, filed Mar. 23, 2016; U.S. Ser. No.62/307,284, filed Mar. 11, 2016; and U.S. Ser. No. 62/173,850, filedJun. 10, 2015, the entire content of each of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates generally to injectable antibiotic formulationsand more specifically to a formulation of an antibiotic macrolidecompound with low toxicity.

Background Information

The present invention is based on the seminal discovery of compositionscontaining an antibiotic macrolide active compound, especiallyazithromycin, formulated for injection to small subjects (about 100pounds or less), such as felines. The formulations of the inventionallow for effective treatment of infections with surprisingly lowertoxicity than other available macrolide formulations.

Azithromycin, for example, is used by veterinarians to treat a range ofbacterial infections in veterinary subjects such as dogs and cats,including streptococci, staphylococci, bartonella henselae, some speciesof chlamydia, haemophilus spp, mycoplasma spp, borrelia burgdorferi andothers. The mechanism of action of azithromycin is binding to the P siteof the 50S ribosomal subunit of those microorganisms that aresusceptible to it, thereby interrupting the microorganism'sRNA-dependent protein synthesis. It is a semi-synthetic macrolideantibiotic derived from erythromycin. Azithromycin is a more popularchoice than erythromycin in the treatment of dogs and cats because ithas a longer half-life and is better absorbed by both species.

However, there are common potential side effects associated withmacrolides such as azithromycin, including gastrointestinal problemslike abdominal discomfort, vomiting and diarrhea. Angioedema andjaundice can also result from taking these drugs. More serious potentialside effects can include cardiac arrhythmia, ventricular tachycardia andissues with renal and hepatic function.

The drug is particularly problematic for use in cats. Azithromycin inparticular is cleared very slowly from feline tissue, resulting indosage schedules that are very convenient, but an increased risk oftoxicity and adverse effects in cats. Given the broad utility ofazithromycin for the treatment of various infections, a need thereforeexists for compositions containing azithromycin that are at least aspotent and effective but have lower toxicity, especially in felines.

SUMMARY OF THE INVENTION

Provided herein is a composition, comprising:

(i) a hydrated form of a macrolide, such as a mectin or mycin,preferably an mono- or di-hydrated macrolide, such as an azilide andmost especially azithromycin;

(ii) a suitable solvent; and

(iii) an excipient.

In certain aspects, the solvent is triacetin. In certain embodiments,the solvent is present in an amount of about 38.0% w/w. In one aspect,the solvent is caprylic/capric triglycerides or caprylic triglycerides.In other embodiments, the triglyceride solvent is present in an amountof about about 54.0% w/w. In some aspects, the composition furthercomprises castor oil, such as KALLIPHOR™ HS15 or RH 40™. In certainaspects, the macrolide is present in an amount of about 7.0% w/w. Inother aspects, the composition is formulated for administration byinjection.

Also provided herein is a method of treating an infection in an animalor a small human subject, generally about 100 pounds or less in weightwith a single injection of a composition of the invention, requiringonly one dose in a single injection for resolution of the infection upto 100%. No additional dosing for the infection treated should berequired (although, of course, re-dosing is possible if a separateinfection occurs).

The animal may be a feline including, but not limited to, a domesticcat. The method provided herein includes administering an effectiveamount of a composition comprising (i) a macrolide such as azithromycin,preferably in a di- or mono-hydrate form; (ii) a suitable solvent; and(iii) an excipient. In some aspects, the method further comprises anadditional antibiotic that is co-administered with the compositionsprovided herein. In other aspects, the compositions are administered byinjection to the feline for the treatment of an infection.

DETAILED DESCRIPTION OF THE INVENTION

The following terms, definitions and abbreviations apply. Abbreviationsused herein have their conventional meaning within the chemical andbiological arts.

The term “patient” refers to organisms to be treated by the methods ofthe disclosure. Such organisms include, but are not limited to, felinessuch as domestic cats. In the context of the disclosure, the term“subject” generally refers to an individual who will receive or who hasreceived treatment described below (e.g., administration of thecompounds of the disclosure, and optionally one or more additionaltherapeutic agents).

The term “therapeutically effective amount” means the amount of thecompound or pharmaceutical composition that will elicit the biologicalor medical response of a patient or tissue that is being sought by theresearcher, veterinarian, medical doctor or other clinician.

By “pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The terms “administration of” and or “administering a” compound shouldbe understood to mean providing a compound of the disclosure orpharmaceutical composition to the subject in need of treatment.

The disclosure also provides pharmaceutical compositions comprising atleast one active compound in an amount effective for treating adisorder, and a pharmaceutically acceptable vehicle or diluent. Theactive compound will be a macrolide antibiotic, including the mectins(including, without limitation, doximectin and abimectin) and the mycins(including, without limitation, roxithromycin, clarithromycin,tulathromycin, gamithromycin, dirithromycin, fidaxomicin, megalomicin,erythromycin and the like), potentially an azilide, and most preferablyazithromycin. The active agents are most preferably hydrated; e.g., amonohydrate or dehydrate form of the molecule. The compositions of thedisclosure may contain other therapeutic agents than azithromycin andmay be formulated, for example, by employing conventional solid orliquid vehicles or diluents, as well as pharmaceutical additives of atype appropriate to the mode of desired administration (for example,excipients, binders, preservatives, stabilizers, flavors, etc.)according to techniques such as those well known in the art ofpharmaceutical formulation.

The compounds of the disclosure may also be formulated into therapeuticcompositions as natural or salt forms. Pharmaceutically acceptablenon-toxic salts include the base addition salts (formed with freecarboxyl or other anionic groups), which may be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like.Such salts may also be formed as acid addition salts with any freecationic groups and will generally be formed with inorganic acids suchas, for example, hydrochloric, sulfuric, or phosphoric acids, or organicacids such as acetic, citric, p-toluenesulfonic, methanesulfonic acid,oxalic, tartaric, mandelic, and the like. Salts of the disclosureinclude amine salts formed by the protonation of an amino group withinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like. Salts of thedisclosure may also include amine salts formed by the protonation of anamino group with suitable organic acids, such as p-toluenesulfonic acid,acetic acid, and the like.

Additional excipients which are contemplated for use in the practice ofthe disclosure are those available to those of ordinary skill in theart, for example, those found in the United States Pharmacopeia Vol.XXII and National Formulary Vol. XVII, U.S. Pharmacopeia Convention,Inc., Rockville, Md. (1989), the relevant contents of which isincorporated herein by reference. In addition, polymorphs, hydrates, andsolvates of the compounds are included in the disclosure, with hydratesbeing particularly preferred. It should be noted that while the hydratemolecules will contribute water to the pharmaceutical composition, it ismost preferred that no other water source be included.

The disclosed pharmaceutical compositions could be administered by anysuitable means, for example, orally, sublingually; buccally;parenterally, such as by subcutaneous, intravenous, intramuscular,intrathecal, or intracisternal injection or infusion techniques (e.g.,as sterile injectable non-aqueous solutions or suspensions); nasallysuch as by inhalation spray; topically, such as in the form of a creamor ointment; or rectally such as in the form of suppositories; in dosageunit formulations containing non-toxic, pharmaceutically acceptablevehicles or diluents. Preferably, however, the administration will be byinjection or infusion; e.g., by intravenous, subcutaneous orintramuscular routes of administration.

The pharmaceutical compositions for the administration of the compoundsof this embodiment either alone or in combination with other agents,e.g., anti-inflammatories, analgesics, other antibiotics, anti-fungals,anti-virals and other pharmaceutically active components, although thecomposition is effective against infection with a hydrated macrolide,preferably an azilide, most preferably azrithromycin as the sole activeagent present.

The composition may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general, the pharmaceutical compositions are prepared byuniformly and intimately bringing the active ingredient into associationwith a carrier suitable for use in an injection. In the pharmaceuticalcomposition the active object compound is included in an amountsufficient to produce the desired effect upon the process or conditionof diseases.

The pharmaceutical compositions is preferably in the form of a sterileinjectable oleaginous solution or suspension. The composition may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above,preferably not including water. The solvent used in the suspension ispreferably one which is miscible with a medium chain triglyceridesurfactant, preferably a C8 trigylceride.

In certain aspects, the solvent is triacetin (glyceryl triacetate orglycerol triacetate). In certain such embodiments, the solvent ispresent in an amount of about 23 to 70%, 30 to 60%, 40 to 55%, 34 to45%, and preferably about 38.0% w/w. In another aspect, the solvent iscaprylic/capric (C10 and/or C8) triglycerides or caprylic (C8)triglycerides, most preferably a C8 triglyceride. In such embodiments,the triglyceride solvent is present in an amount of about 20 to 60%, 40to 55% and preferably about 54.0% w/w. Other suitable solvents may bebenzyl alcohol, 2-ethoxy (2-ethoxy) ethanol, ethyl oleate, ethylacetate, ethanol, ethyl benzoate, benzyl benzoate, 2-pyrrolidone, DMSOand 2-methyl-2pyrrolidone and 2-pyrrolidone.

The composition most preferably includes at least a solvent and asurfactant; most preferably, triacetin and a C8 triglyceride. In someaspects, the composition comprises a surfactant such as castor oil orhydrogenated castor oil, such as KALLIPHOR™ HS15 or RH 40 or TPGS,polysorbate (e.g., 20 and 80) or lecithen. No depot is formed in thecomposition of the invention.

The formulation can also contain other inert ingredients such asantioxidants or preservatives. Antioxidants such as a propyl gallate,BHA (butylated hydroxy anisole), BHT (butylated hydroxy toluene)monothioglycerol, tri-ethyl citrate, citric acid, TBHQ (tert-butylhydroquinone) and the like may be added to the present formulation. Theantioxidants are generally added to the formulation in amounts of fromabout 0.01 to about 2.0% (w/v). Preservatives such as the parabens(methylparaben and/or propylparaben) are suitably used in theformulation in amounts ranging from about 0.01 to about 2.0 w/v.

The formulation of the present invention may be prepared by adding adispersion of hydrogenated castor oil in acetylated monoglycerides,propyl dicaprylates/dicaprates or caprylic/capric triglycerides to asolution comprising the therapeutic agent. Since the formulation isintended for injection, it is desirable that it be sterilized.Surprisingly, heat sterilization may be used in crafting theformulations of the invention without adversely affecting the stabilityor potency of the macrolide therapeutic agent.

In the methods described herein, an appropriate dosage level willgenerally be about 0.01 to about 50 mg/kg, such as, for example, 0.25 toabout 15 mg/kg per day, such as 2.5 to about 14 mg/kg per day. Withinthis range the dosage may be 0.25 to 0.5, 0.25 to 14 mg/kg, 7 to 10mg/kg (including all intermediate dosages, such as 7.1, 7.2, 7.3 etc.mg/kg) and preferably about 7 mg/kg, all in a single injection form. Inthis form, the compounds need only be administered by single injection,one time for an entire course of treatment to clinically resolve aninfection up to 100% elimination.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the patient undergoing therapy.

EXAMPLE 1 Efficacy and Safety

This study evaluated the effectiveness and field safety of a singleinjection of a triglyceride azithromycin formulation for the treatmentof skin and soft tissue infections (abscesses) in cats.

Twenty-two (22) cats were enrolled in the study at 2 study sites. All 22cats were treated with the investigational veterinary product (IVP), and20 were included in the efficacy evaluation.

Cats enrolled in the study presented to the clinic with skin and softtissue infections. On Day 0, a physical examination was conducted, whichincluded assignment of a wound clinical score based on swelling, pain,and exudate. For inclusion in the study, the wound clinical score had tobe a minimum of 5, with a minimum exudate score of 2. A swab wasobtained from the wound (following lancing for closed abscesses) andshipped to each investigator's preferred contract laboratory forbacterial culture. Wound management procedures were allowed after swabcollections, but the only permissible cleaning agents were saline or tapwater. Blood and serum samples were collected, and hematology and serumchemistry analyses were conducted in-clinic.

The cats were dosed via subcutaneous injection with about 7 mg/kg of acomposition consisting of 7% azithromycin DH (dihydrate), 54% caprylic(C8) triglycerides, 38% triacetin and 1% KOLLIPHOR™ HS-15, at which timeobservations were made for injection pain:

Dosing Chart Injection Dose Rate Weight (kg) Weight (lbs) Volume (mL)(mg/kg) 1.5 & 2.0 >3.3 & .5 4.4 0.2 7.0-9.3 >2.0 & .5_ 3.0 >4.4 & 6.60.3  7.0-10.0 >3.0 & 5. 4.0 >6.6 & 8.8 0.4 7.0-9.0 >4.0 & 5.0 >8.8 &11.0 0.5 7.0-8.5 >5.0 & 5_ 6.0 >11.0 & 13.2 0.6 7.0-8.2 >6.0 & 5.7.0 >13.2 & .5 15.4 0.7 7.0-8.0 >7.0 & 8.0 >15.4 & 5 17.6 0.87.0-7.9 >8.0 & 5 9.0 >17.6 & 5 19.8 0.9 7.0-7.8 >9.0 & 5 10.0 >19.8 &22.0 1.0 7.0-7.7

Observations were made hourly for the first 4 hours postinjection, andan injection site observation and temperature were obtained at 4 hourspost-injection. At approximately 24 hours post-injection, anothertemperature and injection site evaluation were conducted.

At the interim study visit on Day 7 (±2), a wound clinical score wasassigned and the injection site was examined for any abnormalities. Atthe final visit on Day 14 (±2), a physical examination was conducted, awound clinical score was assigned, and the injection site was evaluatedfor any abnormalities. In addition, blood and serum samples werecollected for a final hematology and serum chemistry analysis.

A successful case was defined as a cat with a concluding wound score of1 for at least two of the three variables, and an improvement of atleast 1 or a score of 1 in the third variable. Since all cases weresuccessful, there were no Day 14 swab samples obtained for culture.

Based on wound clinical scores, 20 of 20 cats in the effectivenessanalysis were considered successful cases, resulting in an efficacy of100%.

Case D0 D7 D14 Day 14 ID Score Score Score Day 0 Culture Culture ROB01 74 3 Pasteurella species, Enterobacter cloacae, Citrobacter freundii, NA& Pseudomonas species ROB02 8 3 3 Pasteurella species NA ROB03 7 3 3Pasteurella species & non-enteric gram negative rod NA ROB04 7 3 3Pasteurella species & Staphylococcus schleiferi NA ROB05 9 3 3Pasteurella species NA ROB06 9 3 3 Pasteurella species NA ROB07 7 3 3Hemolytic Staphylococcus species & Serratia fonticola NA ROB08 9 3 3Pasteurella species NA ROB09 7 4 3 Pasteurella multocida,alpha-hemolytic Streptococcus sp. & NA Pasteurella species ROB10 7 3 3Pseudomonas putida & Pasteurella species NA ROB11 7 3 3 Pasteurellaspecies & non-enteric gram negative rod NA ROB12 9 4 3 Pasteurellaspecies & non-enteric gram negative rod NA SIF01 7 3 3 Gram positivefusiform anaerobe NA SIF02 6 3 3 No pathogens isolated NA SIF03 9 3 3Staphylococcus aureus NA SIF04 9 3 3 Coagulase negative Staphylococcus &mixed gram positive and NA negative anaerobic reds SIF05 9 3 3 Coagulasenegative Staphylococcus sp. NA SIF06 9 3 3 Gram negative bacilli NA

indicates data missing or illegible when filed

EXAMPLE II Dose Testing

The dose of active described in Example I was halved to determinewhether efficacy could be achieved at a lower dosing concentration. Inparticular, this study evaluated the safety and effectiveness of asingle administration of an azithromycin injectable formulation (IVP)for the treatment of skin infections (wounds and abscesses) in cats whenadministered at two dose levels as compared to a negative control ofsterile saline (CVP).

Twenty-one (21) cats were enrolled in the study at 2 study sites. Seven(7) cats were treated with the IVP at a dose rate of 7 mg/kg (Group C),7 cats were treated with the IVP at a dose rate of 3.5 mg/kg (Group B),and 7 cats were treated with the CVP (Control, Group A). All 21 catswere included in the safety evaluation, while 18 were included in theeffectiveness evaluation.

Cats enrolled in the study presented to the clinic with skin infections.On Day 0, a physical examination was conducted, which includedassignment of a wound clinical score based on swelling, pain, andexudate. For inclusion in the study, the wound clinical score had to bea minimum of 5, with a minimum exudate score of 2. A swab was obtainedfrom the wound (following lancing for closed abscesses) and shipped toeach investigator's preferred contract laboratory for bacterial culture.Wound management procedures were allowed after swab collections, but theonly permissible cleaning agents were saline or tap water.

The cats were dosed via subcutaneous injection and the injection sitewas recorded. At the first interim study visit on Day 3 (−1 day), aphysical examination was conducted, a clinical score was assigned, andthe injection site was examined for abnormalities. If there was noimprovement in the clinical score, or if the exudate score was 3(purulent exudate present), the cat was withdrawn from the study and aswab sample was collected for bacterial culture.

At the second interim study visit on Day 7 (±1 day), a physicalexamination was conducted, a clinical score was assigned, and theinjection was examined for abnormalities. If the case was a treatmentfailure based on the clinical score (success=concluding clinical scoreof 1 for at least two of the three variables, and an improvement of atleast 1 or a score of 1 in the third variable as compared to Day 0clinical score), it was withdrawn from the study and a swab sample wascollected for bacterial culture.

For cats that were treatment successes on Day 7, a final study visit wasconducted on Day 14 (±2 days). A clinical score was assigned and, sincethere were no cases with recurrence, no final swab samples werecollected on Day 14 for bacterial culture.

Based on wound clinical scores, 6 of 6 cats in Group C were consideredsuccessful cases; 6 of 7 cats in Group B were considered successfulcases; and 2 of 5 cats in Group A were considered successful cases.Thus, the efficacy for the azithromycin 7 mg/kg dose was 100.0% versusan efficacy of 85.7% for the 3.5 mg/kg dose and an efficacy of 40.0% forthe control group.

EXAMPLE III Toxicity Evaluation

Dose levels of control, 7 mg/kg One (Day 0) and 35 mg/kg One (Day 0) ofazithromycin active prepared in the composition of Example I wereevaluated for adverse reactions in felines.

Cats were dosed as described in the Examples above via subcutaneous (SQ)injection into the right dorsoscapular area. Injection sites wereevaluated once in acclimation, at four hours post-dosing, and once dailyfrom Days 1 to 7. Rectal temperatures were taken at four hourspost-dosing, once daily post-dosing, then discontinued at three dayspost-dosing as rectal temperatures remained within test facilityreference ranges. Blood was collected for clinical pathology (hematologyand clinical chemistry) from all cats on Study Days −7, 3, and 7.Standard six-lead and rhythm strip electrocardiographs (ECGs) wereobtained from each cat once during acclimation, on Day 0 at two hourspost-dosing, on Day 1 at 24 hours post-dosing, and then on Day 7.

Cats remained in good general health throughout the study. All adverseevents (AEs) observed during the study were non-serious andself-limiting. AEs related to test article administration were asfollows: There were no serious adverse events noted. There were nonotations of erythema, heat, or swelling at injection sites. One cat ingroup T5 was noted to be painful at 4 h post-dosing. The site wasnon-painful by the next scheduled observation.

Minor abnormalities were noted for serum chemistry and hematologyparameters. Elevations noted in creatinine phosphokinase and lactatedehydrogenase were consistent with handling stress during bloodcollection. Several other clinical chemistry parameters were noted to beoutside of test facility reference ranges on multiple cats during thestudy. None of these abnormalities were of clinical or toxicologicalconcern.

Electrocardiographs were reviewed by the cardiologist and showed noevidence of cardiac pathology. Two cats had minor abnormalities on ECG.One cat from group T1 had multiple ventricular premature contractions;and one cat in group T5 had sinus arrhythmia with frequent escape beats.Both of these were noted on the Day 7 ECG. No cardiovascularabnormalities were noted on physical examination of either cat duringacclimation or during the study. Given the lack of other clinical signsof cardiac disease, and the isolated nature of these findings, they haveno clinical or toxicological relevance and are therefore unrelated totest article administration.

Rectal temperatures remained within test facility reference rangesthroughout the entire study. Dose group had no effect on clinicalchemistry, hematology, ECG, rectal temperature, physical examination,body weight, and food consumption outcomes.

In conclusion, azithromycin 7% injection, when administered SQ in catsat 1 and 5 times the proposed label dose versus a placebo control wasnot associated with any clinically or toxicologically relevant effectson clinical chemistry, hematology, ECG, rectal temperature, or foodconsumption.

Although the invention has been described with reference to the abovedescription, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A composition, comprising: (i) a hydratedmacrolide; (ii) at least one suitable solvent; and (iii) an excipient.2. The composition of claim 1, wherein the solvent is triacetin.
 3. Thecomposition of claim 1, wherein the macrolide is azithromycin.
 4. Thecomposition of claim 1, wherein the solvent is present in an amount ofabout 38.0% w/w.
 5. The composition of claim 1, further comprising asurfactant.
 6. The composition of claim 5, wherein the surfactant iscastor oil.
 7. The composition of claim 5, wherein the castor oilcomprises kalliphor HS15.
 8. The composition of claim 1, wherein thesolvent is caprylic or caprylic/capric triglycerides.
 9. The compositionof claim 8, wherein the solvent is present in an amount of about 54.0%w/w.
 10. The composition of claim 1, wherein azithromycin is present inan amount of about 7.0% w/w.
 11. The composition of claim 1, wherein thesolvent is triacetin and caprylic, caprylic or capric triglycerides. 12.The composition of claim 8 or claim 11, wherein the triglyceride is a C8triglyceride.
 13. The composition of claim 1, wherein the composition isformulated for injection.
 14. A method of treating an infection in asubject weighing less than about 100 pounds, comprising administering aneffective amount of a composition of claim
 1. 15. The method of claim14, further comprising administering an additional antibiotic incombination with the composition of claim
 1. 16. The method of claim 14,wherein the subject is a feline.
 17. The method of claim 14, wherein thecomposition is administered by injection.
 18. A composition formulatedfor injection, comprising about 54.0% w/w caprylic triglycerides; about38.0% w/w triacetin; KALLIPHOR™ HS15; and about 7.0% w/w azithromycinDH.